U.S. patent application number 12/742831 was filed with the patent office on 2011-04-21 for generation of an hla-negative osteogenic precursor cell line.
This patent application is currently assigned to OSTEOSPHERE, LLC. Invention is credited to Mark Brinker, Mark S.F. Clarke, Alamelu Sundaresan.
Application Number | 20110091862 12/742831 |
Document ID | / |
Family ID | 40639441 |
Filed Date | 2011-04-21 |
United States Patent
Application |
20110091862 |
Kind Code |
A1 |
Clarke; Mark S.F. ; et
al. |
April 21, 2011 |
GENERATION OF AN HLA-NEGATIVE OSTEOGENIC PRECURSOR CELL LINE
Abstract
The present disclosure provides methods for producing human
osteogenic cell lines in which HLA Class I expression is absent or
reduced. The resulting osteogenic cells can then be used, for
example, to form mineralized three-dimensional bone constructs
which can be used, for example, in bone grafting procedures. The
lack of HLA Class I expression in the cells of the mineralized
three-dimensional bone constructs reduces or eliminates the
potential for host-graft rejection.
Inventors: |
Clarke; Mark S.F.; (Houston,
TX) ; Brinker; Mark; (Houston, TX) ;
Sundaresan; Alamelu; (Sugarland, TX) |
Assignee: |
OSTEOSPHERE, LLC
Houston
TX
|
Family ID: |
40639441 |
Appl. No.: |
12/742831 |
Filed: |
November 13, 2008 |
PCT Filed: |
November 13, 2008 |
PCT NO: |
PCT/US08/83443 |
371 Date: |
December 15, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60988025 |
Nov 14, 2007 |
|
|
|
Current U.S.
Class: |
435/1.1 ;
435/366 |
Current CPC
Class: |
C12N 5/0654 20130101;
C12N 2501/50 20130101; C12N 2525/00 20130101 |
Class at
Publication: |
435/1.1 ;
435/366 |
International
Class: |
C12N 5/077 20100101
C12N005/077 |
Claims
1. A method for producing a human osteogenic cell line in which HLA
Class I expression is silenced, the method comprising: providing
adult mesenchymal stem cells or embryonic mesenchymal stem cells;
introducing into said adult mesenchymal stem cells or said
embryonic mesenchymal stem cells a short hairpin RNA (shRNA)
cassette capable of directing expression of an inhibitory RNA
specific for the HLA Class I gene; and identifying and propagating
individual cells in which the HLA Class I RNA gene is silenced.
2. The method of claim 1 wherein said shRNA cassette is introduced
into said adult mesenchymal stem cells or said embryonic
mesenchymal stem cells using impact-mediated loading.
3. A human osteogenic line produced by a method comprising:
providing adult mesenchymal stem cells or embryonic mesenchymal
stem cells; introducing into said adult mesenchymal stem cells or
said embryonic mesenchymal stem cells a short hairpin RNA (shRNA)
cassette capable of directing expression of an inhibitory RNA
specific for the HLA Class I gene; and identifying and propagating
individual cells in which the HLA Class I RNA gene is silenced.
4. The human osteogenic line of claim 3 wherein said shRNA cassette
is introduced into said adult mesenchymal stem cells or said
embryonic mesenchymal stem cells using impact-mediated loading.
5. A method for producing a human osteogenic cell line in which HLA
Class I expression is silenced, the method comprising: providing
adult mesenchymal stem cells or embryonic mesenchymal stem cells;
introducing into said adult mesenchymal stem cells or said
embryonic mesenchymal stem cells a DNA specific for the HLA Class I
gene wherein said DNA disrupts said HLA Class I gene as a result of
site specific recombination; and identifying and propagating
individual cells in which the HLA Class I gene is silenced.
6. The method of claim 5 wherein said DNA specific for the HLA
Class I gene is introduced into said adult mesenchymal stem cells
or said embryonic mesenchymal stem cells using impact-mediated
loading.
7. A human osteogenic line produced by a method comprising:
providing adult mesenchymal stem cells or embryonic mesenchymal
stem cells; introducing into said adult mesenchymal stem cells or
said embryonic mesenchymal stem cells a DNA specific for the HLA
Class I gene wherein said DNA disrupts said HLA Class I gene as a
result of site specific recombination; and identifying and
propagating individual cells in which the HLA Class I gene is
silenced.
8. The human osteogenic line of claim 7 wherein said DNA specific
for the HLA Class I gene is introduced into said adult mesenchymal
stem cells or said embryonic mesenchymal stem cells using
impact-mediated loading.
9. A method for producing mineralized three-dimensional bone
constructs, the method comprising: providing a human osteogenic
cell line in which HLA Class I expression is silenced;
differentiating cells in said human osteogenic cell line into
osteoblasts and osteoclast precursors; introducing said osteoclast
precursors and said osteoblasts into a cylindrical culture vessel
that rotates about a central horizontal axis, said cylindrical
culture vessel comprising a matrix-free culture medium; culturing
said osteoblasts and said osteoclast precursors in said cylindrical
culture vessel during horizontal rotation at a rate effective to
create low shear conditions and to promote the formation of
aggregates comprising said osteoclast precursors and said
osteoblasts; further culturing said aggregates in said cylindrical
culture vessel during horizontal rotation at a rate effective to
create low shear conditions, whereby said aggregates grow in size
and said osteoclast precursors differentiate into osteoclasts;
introducing a matrix-free mineralization culture medium into said
cylindrical culture vessel and culturing said aggregates during
horizontal rotation at a rate effective to create low shear
conditions, wherein mineralized three-dimensional bone constructs
are formed.
10. The method of claim 9 wherein providing said human osteogenic
cell line comprises: providing adult mesenchymal stem cells or
embryonic mesenchymal stem cells; introducing into said adult
mesenchymal stem cells or said embryonic mesenchymal stem cells a
short hairpin RNA (shRNA) cassette capable of directing expression
of an inhibitory RNA specific for the HLA Class I gene; and
identifying and propagating individual cells in which the HLA Class
I RNA gene is silenced.
11. The method of claim 10 wherein said shRNA cassette is
introduced into said adult mesenchymal stem cells or said embryonic
mesenchymal stem cells using impact-mediated loading.
12. The method of claim 9 wherein providing said human osteogenic
cell line comprises: providing adult mesenchymal stem cells or
embryonic mesenchymal stem cells; introducing into said adult
mesenchymal stem cells or said embryonic mesenchymal stem cells a
short hairpin RNA (shRNA) cassette capable of directing expression
of an inhibitory RNA specific for the HLA Class I gene; and
identifying and propagating individual cells in which the HLA Class
I RNA gene is silenced.
13. The method of claim 12 wherein said shRNA cassette is
introduced into said adult mesenchymal stem cells or said embryonic
mesenchymal stem cells using impact-mediated loading.
14. A mineralized three-dimensional bone construct produced by a
method comprising: providing a human osteogenic cell line in which
HLA Class I expression is silenced; differentiating cells in said
human osteogenic cell line into osteoblasts and osteoclast
precursors; introducing said osteoclast precursors and said
osteoblasts into a cylindrical culture vessel that rotates about a
central horizontal axis, said cylindrical culture vessel comprising
a matrix-free culture medium; culturing said osteoblasts and said
osteoclast precursors in said cylindrical culture vessel during
horizontal rotation at a rate effective to create low shear
conditions and to promote the formation of aggregates comprising
said osteoclast precursors and said osteoblasts; further culturing
said aggregates in said cylindrical culture vessel during
horizontal rotation at a rate effective to create low shear
conditions, whereby said aggregates grow in size and said
osteoclast precursors differentiate into osteoclasts; introducing a
matrix-free mineralization culture medium into said cylindrical
culture vessel and culturing said aggregates during horizontal
rotation at a rate effective to create low shear conditions,
wherein mineralized three-dimensional bone constructs are
formed.
15. The mineralized three-dimensional bone construct of claim 14,
wherein providing said human osteogenic cell line comprises:
providing adult mesenchymal stem cells or embryonic mesenchymal
stem cells; introducing into said adult mesenchymal stem cells or
said embryonic mesenchymal stem cells a short hairpin RNA (shRNA)
cassette capable of directing expression of an inhibitory RNA
specific for the HLA Class I gene; and identifying and propagating
individual cells in which the HLA Class I RNA gene is silenced.
16. The method of claim 15 wherein said shRNA cassette is
introduced into said adult mesenchymal stem cells or said embryonic
mesenchymal stem cells using impact-mediated loading.
Description
CLAIM OF PRIORITY
[0001] The present application for patent claims priority to U.S.
Provisional Application No. 60/988,025 entitled "Generation of an
HLA-Negative Osteogenic Precursor Cell Line" filed Nov. 14, 2007,
assigned to the assignee hereof, and hereby expressly incorporated
by reference herein.
FIELD OF THE INVENTION
[0002] The invention relates to the preparation of human osteogenic
cell lines which may be used to prepare ex vivo-derived mineralized
three-dimensional bone constructs which replicate natural bone.
BACKGROUND
[0003] One of the central problems associated with studying both
the normal and pathophysiology of bone is that as an organ system
it is slow growing and the time to show an observable response to a
particular stimulus is relatively long. The nature of the
mineralized tissue matrix of bone in vivo and its complex
architecture also presents several technical problems associated
with how experimental observations can be made. At present, truly
informative studies designed to understand bone physiology have
relied primarily on the removal of samples of bone tissue from
normal or diseased tissue either in a clinical setting or from
experimental animal models.
[0004] To date, there is no three dimensional tissue culture model
of bone, either of animal or human origin. The prior art has relied
primarily on the use of monotype cell type cultures of osteoblasts
or osteoclast cells grown on planar, two dimensional tissue culture
surfaces. Such cultures have also been grown in three dimensional
collagen support gels and some investigators have utilized culture
systems that allow types of mechanical strain to be applied to the
cells in order to study the effects of mechanical loading. However,
these cultures have been primarily focused on the responses of a
single cell type, such as osteoblasts, to various environmental
stimuli.
[0005] Existing planar monotype tissue culture models of bone do
not allow the study of the interactions between the different cell
types present in normal bone responsible for normal bone
remodeling. The developmentally inactive osteocyte cell type
present in the mineralized matrix of normal bone in vivo (from
which osteoblasts are derived) have yet to be fully characterized
in any tissue culture model due to their supposed transformation
into osteoblasts once they have been removed from the bone matrix
and placed into culture.
[0006] Moreover, the process of mineralization, which is essential
to the formation of new bone, has previously only been studied in
monotype cultures of osteoblasts. The mineralization process has
been studied in such models in the absence of the major cell type
involved in the removal of mineralized material, namely the
osteoclast. However, the complex interplay between both of these
cell types is essential for normal bone remodeling (i.e. bone
formation and bone loss). Without both cell types being present, a
true in vitro/ex vivo representation of the normal or indeed
pathological processes involved in the bone remodeling process is
impossible. As such, the use of such monotype culture models to
investigate the effects of manipulations, such as anti-osteoporotic
drugs or mechanical load interventions, have limited utility due to
the lack of similarity to the true physiological state existing
within bone tissue in vivo.
SUMMARY
[0007] In one aspect, the disclosure provide methods for producing
a human osteogenic cell line in which HLA Class I expression is
silenced. In one embodiment, the method comprises providing adult
mesenchymal stem cells or embryonic mesenchymal stem cells;
introducing a short hairpin RNA (shRNA) cassette capable of
directing expression of an inhibitory RNA specific for the HLA
Class I gene into the adult mesenchymal stem cells or the embryonic
mesenchymal stem cells; and identifying and propagating individual
cells in which HLA Class I RNA is silenced. The resulting
osteogenic cells can then be used, for example, to form mineralized
three-dimensional bone constructs which can be used, for example,
in bone grafting procedures. The lack of HLA Class I expression in
the cells of the mineralized three-dimensional bone constructs
reduces or eliminates the potential for host-graft rejection.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 is a flowchart of an example of a method for
preparing mineralized three-dimensional bone constructs.
[0009] FIGS. 2A-2B present images of mineralized three-dimensional
bone constructs at 14 days of mineralization (FIG. 2A) and 21 days
of mineralization (FIG. 2B). The scale bars each represent 1
cm.
[0010] FIGS. 3A-3B present images of mineralized three-dimensional
bone constructs. FIG. 3A presents a fluorescence confocal
microscopy image of an optical section through bone constructs in
which the osteoclast precursor cells were labeled with a
fluorescent cell tracking dye (observable as white spots in FIG.
3A). FIG. 3B shows the same constructs viewed in incident laser
light (i.e. non-fluorescent illumination) to illustrate the shape
of the constructs. The scale bar in each of FIG. 3A and FIG. 3B is
200 .mu.m.
[0011] FIG. 4 presents a three dimensional reconstruction of a
large bone construct using Z series confocal imaging. Osteoclast
precursors were labeled with a fluorescent cell tracking dye.
Panels A-I in FIG. 4 are the individual images used by the confocal
imaging software to build the optical reconstruction of the bone
construct in three dimensions, each image representing a sequential
view over the surface of the construct (white spots indicate
individual labeled osteoclast cells). Panel J is a single image of
the surface of a large bone construct in which structures
reminiscent of resorption pits or lacunae found in actively
remodeling bone in vivo can be clearly seen formed by labeled
osteoclasts on the surface of the OsteoSphere (indicated by arrows,
Bar equals 300 microns).
[0012] FIGS. 5A-5D show Alizarin red S staining and von Kossa
staining of sections through a bone construct. FIG. 5A shows a
5.times. magnification image of Alizarin red S staining and FIG. 5B
shows a 20.times. magnification image of Alizarin red S staining
(which appears as the dark regions of the images). FIG. 5C shows a
5.times. magnification image of von Kossa staining, and FIG. 5D
shows a 20.times. magnification image of von Kossa staining (which
appears as the dark regions of the images). FIG. 5E shows a
composite low power image of a complete 10 micron thick frozen
cross-section of a Bouin's fixed OsteoSphere stained with Alizarin
red S.
[0013] FIGS. 6A-6B show Harris Hematoxylin staining of sections
through a bone construct. FIG. 6A is a 5.times. magnification image
and FIG. 6B is a 20.times. magnification image. The dark regions of
the image indicate staining Arrows in FIG. 6B point to large
numbers of cells embedded within the crystalline matrix in the
three dimensional construct.
[0014] FIGS. 7A-7C show images of bone construct in which
osteoclast precursors were labeled with a fluorescent cell tracking
dye prior to formation of the bone construct, and the bone
construct was stained with a primary antibody against osteocalcein
(a marker of osteoblast differentiation) and an Alexa 488-labeled
secondary antibody. FIG. 7A shows osteocalcein staining, FIG. 7B
shows CellTracker-Orange staining, and FIG. 7C shows the same
construct illuminated with incident laser light. The results
indicate that osteocalcein staining and cell tracking dye (both
visible as a white "ring" around the construct in FIGS. 7A and 7B)
are spatially localized to the same area of the construct.
[0015] FIG. 8 shows the results of a real-time quantitative PCR
assay analysis of mRNA extracted from mineralized bone construct
material.
[0016] FIG. 9 depicts an impact-mediated loading apparatus known as
the G-loader.
[0017] FIG. 10 shows fluorescent micrographs demonstrating
enhancement of impact-mediated loading of IgG (fluorescently
labeled) into Swiss 3T3 cells under hypergravity conditions. Panel
A shows control cells (no impact-mediated loading), Panel B shows
impact-mediated loading at 1.times.g, and Panel C shows
impact-mediated loading at 200.times.g.
[0018] FIG. 11 shows fluorescent micrographs of cells that
experienced impact-mediated loading at 200.times.g of a plasmid
construct encoding for a green fluorescent protein. The cells used
were primary human skeletal myoblasts (HSKMC), Swiss 3T3 cells
(3T3) and primary bovine capillary endothelial cells (BCEC).
DETAILED DESCRIPTION
[0019] In one aspect, the present disclosure provides mineralized
three-dimensional bone constructs (sometimes referred to as
"OsteoSpheres" or simply as "bone constructs"). The mineralized
three dimensional constructs of the disclosure are "bone like" in
appearance by visual inspection, in certain important respects
resembling trabecular bone (also known in the art as "spongy
bone"). In preferred embodiments, the mineralized three-dimensional
bone constructs of the disclosure are macroscopic in size and are
approximately spheroidal in shape, preferably between about 200
.mu.m and about 4 mm in diameter; however, larger and smaller bone
constructs are specifically contemplated.
[0020] The bone constructs comprise an inner core surrounded by an
outer layer. The inner core comprises a three-dimensional
crystalline matrix that stains positively with Alizarin Red S stain
and with the von Kossa histochemical stain, indicating that it
comprises mineral elements observed in normal human bone in vivo,
including calcium, phosphates, and carbonates. The inner core also
comprises osteoblasts and/or osteocytes embedded within the
crystalline matrix, and is preferably devoid of necrotic tissue.
Osteocytes are developmentally inactive cells found only in native
bone tissue in vivo and are believed to be formed from osteoblasts
that have become trapped in the crystalline matrix. The outer layer
is comprised of osteoclasts. The cell types in the bone constructs
of the disclosure can be obtained from any mammalian species, but
are preferably obtained from humans.
[0021] In another aspect, the disclosure provides methods for
producing the mineralized three-dimensional bone constructs. In
general, the bone constructs of the disclosure are produced by
culturing osteoclast precursors and osteoblasts together under
randomized gravity vector conditions (approaching those conditions
that cultured cells experience during microgravity culture) in a
matrix-free culture medium. Osteoclast precursors may be obtained
from bone marrow and/or peripheral blood lymphocytes by techniques
well known in the art. Osteoclast precursors may also be obtained
from commercial sources (for example, from Cambrex/Lonza, Inc.).
Osteoblasts, preferably primary human osteoblasts, may also be
obtained by techniques well known in the art, and may also be
obtained from commercial sources (for example, from PromoCell, Inc.
and from Cambrex/Lonza, Inc.). A "matrix-free culture medium" is a
cell culture medium which does not include carrier material (such
as microcarrier beads or collagen gels) onto which osteoblasts and
osteoclast precursors can attach. Suitable cell culture media
include Eagle's Minimal Essential Medium (EMEM) or Dulbecco's
Modified Eagle's Medium (DMEM), preferably supplemented with fetal
bovine serum (FBS). Preferably, the matrix-free culture medium also
comprises osteoblast growth supplements such as ascorbic acid. The
matrix-free culture medium preferably also further comprises
osteoclast differentiation factors, such as Receptor Activator of
NF-kB (RANK) ligand and macrophage colony stimulating factor
(M-CSF). For example, in one embodiment the matrix-free culture
medium comprises FBS-supplemented DMEM, ascorbic acid, RANK ligand,
and M-CSF. Example 2 includes a description of one suitable
matrix-free culture medium.
[0022] The osteoclast precursors and the osteoblasts are cultured
together under randomized gravity vector conditions effective to
achieve the formation of mixed aggregates of the two cell types.
The aggregates are then further cultured under randomized gravity
vector conditions to increase the aggregates size and to
differentiate the osteoclast precursors into mature
osteoclasts.
[0023] After a predetermined time, the aggregates are cultured
under randomized gravity vector conditions in a matrix-free
mineralization culture medium. A "matrix-free mineralization
culture medium" is a cell culture medium that includes one or more
mineralization agents, such as osteoblast differentiation factors,
that induce osteoblasts to produce crystalline deposits (comprising
calcium, phosphate, and carbonates) but which does not include
carrier material (such as microcarrier beads and collagen gels)
onto which osteoblasts and osteoclast precursors can attach. For
example, in one embodiment, a matrix-free mineralization culture
medium comprises FBS-supplemented EMEM or DMEM, supplemented with
the osteoblast differentiation factors. Osteoblast differentiation
factors include beta-glycerophosphate and
hydrocortisone-21-hemisuccinate. Preferably, the matrix-free
mineralization culture medium also includes osteoclast
differentiation factors such as RANK ligand and M-CSF, and also
includes osteoblast growth supplements such as ascorbic acid. For
example, in one embodiment the matrix-free mineralization culture
medium comprises FBS-supplemented DMEM, beta-glycerophosphate,
ascorbic acid, hydrocortisone-21-hemisuccinate, RANK ligand and
M-CSF. Example 2 includes a description of one suitable matrix-free
mineralization medium.
[0024] In preferred embodiments, randomized gravity vector
conditions are obtained by culturing osteoclast precursors and
osteoblasts in a low shear stress rotating bioreactor. Such
bioreactors were initially designed to mimic some of the physical
conditions experienced by cells cultured in true microgravity
during space flight. In general, a low shear stress rotating
bioreactor comprises a cylindrical culture vessel. One or more
ports are operatively associated with the lumen of the vessel for
the introduction and removal of cells and culture media. The
cylindrical culture vessel is completely filled with a culture
medium to eliminate head space. The cylindrical culture vessel
rotates about a substantially central horizontal axis. The
resulting substantially horizontal rotation occurs at a rate chosen
so that (1) there is essentially no relative motion between the
walls of the vessel and the culture medium; and (2) cells remain in
suspension within a determined spatial region of the vessel such
that they experience a continuous "free fall" through the culture
medium at terminal velocity with low shear stress and low
turbulence. This free fall state may be maintained continuously for
up to several months in some applications described in the prior
art. The continuous orbital movement of the medium relative to the
cells also allows for highly efficient transfer of gases and
nutrients.
[0025] In some embodiments, the diameter of the cylindrical culture
vessel is substantially greater than its height. Such cylindrical
culture vessels are often referred to in the art as High Aspect
Ratio Vessels (HARVs). For example, a HARV having a volume of 10 mL
may have a diameter of about 10 cm and a height of about 1 cm. At
least a portion of the vessel walls may be comprised of a gas
permeable membrane to allow gas exchange between the culture medium
and the surrounding incubator environment. A suitable HARV is
described in, for example, U.S. Pat. No. 5,437,998, incorporated by
reference herein in its entirety. One commercial embodiment of a
HARV is the Rotating Cell Culture System (RCCS) available from
Synthecon, Inc.
[0026] In some embodiments, the diameter of the cylindrical culture
vessel is substantially smaller than its height. Such cylindrical
culture vessels are often referred to in the art as Slow Turning
Lateral Vessels (STLVs). STLVs typically have a core, comprised of
a gas permeable membrane, running through the center of the
cylinder in order to allow gas exchange between the culture medium
and the surrounding incubator environment. STLVs are available from
Synthecon, Inc.
[0027] The use of low shear stressing rotating bioreactor culture
systems is described in, for example, Nickerson et al., Immunity.
69:7106-7120 (2001); Carterson et al., Infection & Immunity.
73(2):1129-40 (2005); and in Goodwin et al. U.S. Pat. No.
5,496,722, each of which is specifically incorporated herein by
reference in its entirety.
[0028] In one embodiment, osteoclast precursors and osteoblasts are
introduced into a cylindrical culture vessel in matrix-free culture
medium. The osteoclast precursors and the osteoblasts may be
introduced into the cylindrical culture vessel separately, or they
may be introduced into the cylindrical culture vessel as a
pre-mixture of the two cell types. Preferably, the cells are
introduced into the cylindrical culture vessel at a
osteoblast:osteoclast precursor ratio of from about 2:1 to about
3:1, although higher and lower ratios are within the scope of the
disclosure. The absolute number of cells introduced into the
cylindrical culture vessel may also be varied. For example, in some
embodiments where a ratio of about 2:1 is employed, about 2 million
osteoblasts and about 1 million osteoclast precursors are
introduced; in other embodiments about 4 million osteoblasts and
about 2 million osteoclast precursors are introduced; and in still
further embodiments about 8 million osteoblasts and about 4 million
osteoclast precursors are introduced. The ratio of
osteoblasts:osteoclast precursors and the absolute number of cells
can be varied in order to vary the size and the number of
aggregates formed. In addition, other cell types may also be
introduced into the cylindrical culture vessel. For example, bone
marrow stroma and stem cells may be cultured along with the
osteoblasts and the osteoclast precursors.
[0029] One or more cell types may optionally be labeled with a
cell-tracking marker, such as a fluorescent cell-tracking dye,
prior to their introduction into the cylindrical culture vessel. In
this way, it is possible to determine the location of the
individual cell types during, or at the conclusion of, the
formation of the bone constructs. For example, fluorescent
CellTracker dyes, available from Invitrogen, Inc., may be used in
conjunction with fluorescence microscopy techniques, such as
confocal fluorescence microscopy. If more than one cell type is
labeled, then they are labeled with different colored dyes so that
each cell type can be tracked independently.
[0030] Cells are then cultured in the matrix-free culture medium in
the cylindrical culture vessel during substantially horizontal
rotation to form aggregates of the two cell types. The rate of
substantially horizontal rotation during the aggregation phase is
chosen so that both (1) low shear conditions are obtained; and (2)
the osteoclast precursors and the osteoblasts are able to coalesce
and form aggregates. The rate of substantially horizontal rotation
may be selected by monitoring the cylindrical culture vessel and by
monitoring the cells and aggregates in the cylindrical culture
vessel (for example using microscopy), to insure that the cells and
aggregates are not sedimenting (which may be caused by too low a
rate of rotation) or experiencing mechanical or excessive
hydrodynamic shear stress. In embodiments in which a HARV is used,
osteoclast precursors and osteoblasts may form a "boundary" layer
situated in the middle of the HARV during the aggregation
phase.
[0031] Preferably, the rate of substantially horizontal rotation
during the aggregation phase is lower than the rate typically used
for culturing cells. For example, in embodiments where the
cylindrical culture vessel is a 10 mL HARV having a diameter of
about 10 cm and a height of about 1 cm, substantially horizontal
rotation at less than about 14 revolutions per minute (rpm) may be
used. More preferably, substantially horizontal rotation at less
than about 12 rpm is used. In certain preferred embodiments,
substantially horizontal rotation at between about 1 rpm and about
4 rpm is used. In one specific embodiment, substantially horizontal
rotation at about 2 rpm is used. Note that the aforementioned rpm
values are provided with reference to a 10 mL HARV having the
aforementioned dimensions. The rpm values will vary depending on
the volume and dimensions of the cylindrical culture vessel. The
rpm values during the aggregation phase for all such vessels are
easily determined using the aforementioned methodology.
[0032] Without being bound by a particular theory or mechanism, it
is believed that the use of a matrix-free culture medium allows the
use of rates of rotation that are substantially lower than
previously reported in the art for culturing mammalian cells in a
low shear stress rotating bioreactor. The use of low rotation
rates, in turn, is believed for the first time to promote efficient
association of osteoclast precursors and osteoblasts into
aggregates, and to promote three-dimensional organization of these
two cell types within the aggregates. Thus, the organization of the
cell types within the aggregate is not constrained or influenced by
an exogenous carrier material, but rather by native cell-cell
interaction. Consequently, the three-dimensional organization of
the osteoblasts and osteoclasts is physiologically realistic.
[0033] The rate of substantially horizontal rotation may optionally
be adjusted periodically during the aggregation phase in order to
compensate for the increase in the sedimentation velocity (which is
a function of volume and density) of the forming aggregates,
thereby maintaining the aggregates in low shear "free fall" and
preventing impact with the vessel wall.
[0034] The aggregation phase proceeds for a period of time
sufficient to produce the desired size of aggregates. Aggregate
formation may be monitored during the aggregation phase by visual
inspection, including through the use of microscopy. It will be
apparent from the disclosure that the size of the aggregates is
also dependent on the number of cells that are initially introduced
into the cylindrical culture vessel, the length of time allowed for
aggregation, as well as the rotation rate. In one example, the
aggregation phase is allowed to proceed for between about 24 hours
and about 48 hours.
[0035] Once aggregates of the desired size have formed, the
aggregates are preferably further cultured in the cylindrical
culture vessel during substantially horizontal rotation for a
period of time sufficient to allow the aggregates to grow to a
desired size through cell proliferation and/or to allow the
osteoclast precursors in the aggregates to differentiate into
osteoclasts. For example, the further culturing of the aggregates
may proceed for between about 5 and about 7 days and may lead to
grown aggregates having a diameter from between about 200 .mu.m and
about 4 mm. The resultant aggregates are sometimes referred to
herein as "spheroids." Preferably, the rate of substantially
horizontal rotation during the further culturing is higher than the
rate during the aggregation phase, but still provides low shear
conditions in the cylindrical culture vessel. For example, a
rotation rate of between about 9 rpm and about 16 rpm, preferably
about 14 rpm, may be used during further culturing for the 10 mL
HARV exemplified above. The rate of substantially horizontal
rotation may optionally be adjusted periodically during the further
culturing phase in order to compensate for the increase in the
sedimentation pathway of the aggregates as they grow in size (and
hence undergo changes in volume and density), thereby maintaining
the growing aggregates in low shear "free fall" and preventing
impact with the vessel wall.
[0036] Once aggregates have attained a desired size, a matrix-free
mineralization culture medium is introduced into the cylindrical
culture vessel and the aggregates are cultured during substantially
horizontal rotation until they become mineralized (either partially
mineralized or fully mineralized), thereby forming the mineralized
three-dimensional bone constructs of the disclosure. For example,
the mineralization process may proceed for between about 7 days and
about 21 days depending on the size of the aggregates and the
degree of mineralization required. Preferably, the rate of
substantially horizontal rotation during such the mineralization
process is higher than the rate during the aggregation phase, but
still provides low shear conditions in the cylindrical culture
vessel. For example, a rotation rate of between about 9 rpm and
about 20 rpm, preferably about 14 rpm, may be used during the
mineralization phase for the 10 mL HARV exemplified above. The rate
of substantially horizontal rotation may optionally be adjusted
periodically during the mineralization phase in order to compensate
for the increase in the sedimentation pathway of the aggregates as
they increase in mass, thereby maintaining the mineralizing
aggregates in low shear "free fall" and preventing impact with the
vessel walls.
[0037] Mineralized three-dimensional bone constructs are harvested
once they have achieved the desired size and mass. In cylindrical
culture vessels with one or more access ports, the bone constructs
are removed through a part. When the bone constructs exceed the
diameter of the port, the vessel is disassembled to remove the bone
constructs.
[0038] Osteoclasts and osteoblasts act coordinately in the
mineralization process that occurs in vivo during bone formation
and bone restructuring. Accordingly, the mineralized
three-dimensional bone constructs of the disclosure, formed by the
coordinated activity of osteoblasts and osteoclasts, are
physiologically realistic.
[0039] As described above, the mineralized three-dimensional bone
constructs of the disclosure mimic trabecular bone in many
important aspects. The bone constructs of the disclosure therefore
have a great many uses in the fields of, for example, physiology
research and development, pharmaceutical research, and orthopedics.
Without limitation, these include the direct benefit of developing
a model for studying both normal bone physiology and the
pathological responses observed in disease states such as
osteoporosis, as well as providing a highly economical platform for
drug development as it relates to the treatment of bone
diseases.
[0040] The bone constructs of the disclosure also can be used for
autologous grafts. Specifically, diseased or missing bone may be
replaced with ex-vivo-derived mineralized three-dimensional bone
constructs in which the component osteoclasts and osteoblasts are
harvested from healthy bone and peripheral blood lymphocytes of the
patient requiring the bone graft. Examples of pathologies where the
bone constructs of the disclosure have therapeutic utility include
fractures, non-unions of fractures, congenital deformities of bone,
bone infections, bone loss, segmental bone defects, bone tumors,
metabolic and endocrine disorders affecting bone, and tooth
loss.
[0041] The bone constructs of the disclosure can also be used for
allogenic (allograft) grafts. Specifically, diseased or missing
bone can be replaced with ex vivo-derived mineralized
three-dimensional bone constructs in which the component
osteoclasts and osteoblasts are harvested from healthy bone and
peripheral blood lymphocytes of another donor for the benefit of a
patient requiring bone graft. Examples of pathologies where the
bone constructs of the disclosure have therapeutic utility include
fractures, non-unions of fractures, congenital deformities of bone,
bone infections, bone loss, segmental bone defects, bone tumors,
metabolic and endocrine disorders affecting bone, and tooth
loss.
[0042] Because the bone constructs of the disclosure closely
resemble bone formed in vivo, it is expected that they produce
unique factors and/or cytokines essential for bone remodeling.
Accordingly, the bone constructs of the disclosure serve as a
source for identification and harvesting of these factors.
[0043] The bone constructs of the disclosure may also be used to
study the interface between prosthetic devices/materials and bone
tissue.
[0044] Sensors or stimulation devices may be incorporated into the
bone constructs of the disclosure, and the resulting constructs
implanted into bone tissue in vivo.
[0045] The bone constructs of the disclosure also may be used in
the production of large structures of specific dimensions for
"form-fitted" applications such as replacement of large regions of
the skeleton. This may be achieved using a combination of tissue
scaffolding/synthetic support materials embedded with numerous bone
constructs to generate a much larger composite tissue
aggregate.
[0046] The bone constructs of the disclosure also provide a low
cost alternative in which to study the effects of microgravity, and
of other space environment insults, such as radiation, on the
process of bone formation/bone loss.
[0047] The following examples are not to be construed as limiting
the scope of the invention disclosed herein in any way.
EXAMPLES
Example 1
Flow Chart of a Method for Producing Bone Constructs
[0048] A flow chart of the method for producing mineralized
three-dimensional bone constructs is provided in FIG. 1. Osteoblast
and osteoclast precursor cells are first isolated (110) from a
healthy patient and then inoculated (120) into a modified High
Aspect Rotating Vessel (HARV) with a matrix-free culture medium.
Cells are allowed to aggregate (130) at a rotation speed (typically
2 rpm) much lower than that commonly used for the culture of
mammalian cells. Low speed promotes aggregation of the two or more
cell types in the early stages of aggregate formation. After the
aggregation period is over, the rotation speed of the High Aspect
Rotating Vessel is increased (140). This allows the bone construct
to grow into spheroids (150) in a state of "free fall". The
mineralization step (160) is then initiated by exchanging the
initial matrix-free culture medium for a matrix-free mineralization
culture medium, which initiates the production of a calcified
crystalline matrix in the center of the tissue aggregate. The bone
constructs are then characterized. The spatial arrangement of the
different cell types is observed by confocal microscopy imaging
(170). The cells are visualized with Z-series confocal imaging
(175) by pre-labeling the initial cell constituents of the
construct with green fluorescent cell tracker probe. The presence
of calcium, phosphate and carbonate is revealed by using Alizarin
red S stain and Kossa histochemical stain (180), while the presence
of nucleated cells embedded in the crystalline matrix is revealed
by nuclear staining (185). Immuno-staining of the construct (190)
shows that cell markers such as alkaline phosphate are absent from
the cells embedded in the crystalline matrix. Finally, prelabeling
of the osteoclast precursor cells with Cell Tracker-Orange (195)
shows that precursor cells allowed to aggregate and organize under
these culture conditions co-localize with those cells expressing
the osteoblast differentiation marker, namely osteocalcein, as a
surface layer of the OsteoSphere.
Example 2
Production of Bone Constructs in a HARV
[0049] Cryopreserved primary normal human osteoblast cells and
normal human osteoclast precursor cells were purchased from the
Cambrex Corporation (East Rutherford, N.J.) and stored frozen under
liquid nitrogen until needed.
[0050] Osteoblast cells were rapidly thawed by placing the vial in
a 37.degree. C. oven, removing the cell suspension from the vial
and placing it in a 15 ml centrifugation tube and then diluting the
cell suspension with 10 ml of Dulbecco's Modified Essential Medium
(DMEM) supplemented with 10% (v/v) fetal bovine serum (10%
FBS-DMEM). The cells were then collected by centrifugation at
100.times.g for 5 min at 4.degree. C. The supernatant was then
removed and the cell pellet was resuspended by gentle tituration in
10 ml of fresh 10% FBS-DMEM supplemented with 5 .mu.M ascorbic acid
and 1 mg/ml GA-1000 (gentamicin/amphotericin B mixture). This
process was carried out to wash away the cryopreservatives in which
the osteoblast cells had been frozen.
[0051] The resulting cell suspension was then inoculated into a
T-75 tissue culture flask and incubated at 37.degree. C. in a 5%
CO.sub.2 atmosphere tissue culture incubator for a total period of
seven days, with the medium being exchanged every three days. After
seven days the osteoblast culture was approaching confluence and
the osteoblast cells were harvested by removing the cells from the
surface of the flask using trypsin/EDTA digestion followed by
collection of the cells by centrifugation as above. The cell pellet
was then gently resuspended in 20 ml of fresh 10% FBS-DMEM
supplemented with 5 .mu.M ascorbic acid and 1 mg/ml GA-1000. The
resulting cell suspension was then inoculated into two T-75 tissue
culture flasks and again cultured for an additional seven days.
This process of osteoblast cell expansion continued until the cells
had reached passage 5 (i.e. five expansion/population doubling
cycles).
[0052] When the osteoblast cells had reached Passage 5 in culture
they were harvested using trypsin/EDTA digestion followed by
collection of the cells by centrifugation as above. The cell pellet
was then gently resuspended in 10 ml of fresh 10% FBS-DMEM
supplemented with 5 .mu.M ascorbic acid, 100 U/ml penicillin and
100 ug/ml streptomycin, penicillin/streptomycin being substituted
for GA-1000 at this point due to the potential negative effects of
gentamicin on the capability of osteoblast cells to produce
mineralized extracellular matrix. The resulting osteoblast cell
suspension was counted using a hemacytometer to ascertain the
number of osteoblast cells/ml. An aliquot of cell suspension
containing a total of six million osteoblast cells was removed and
placed in a separate 15 ml centrifugation tube in preparation for
the addition of osteoclast precursor cells.
[0053] Osteoclast precursor cells were rapidly thawed by placing
the vial in a 37.degree. C. oven, removing the cell suspension from
the vial and placing it in a 15 ml centrifugation tube and then
diluting the cell suspension with 10 ml of Dulbecco's Modified
Essential Medium (DMEM) supplemented with 10% (v/v) fetal bovine
serum (10% FBS-DMEM). The cells were then collected by
centrifugation at 100.times.g for 5 min at 4.degree. C. The
supernatant was then removed and the cell pellet was resuspended by
gentle tituration in 1 ml of fresh 10% FBS-DMEM supplemented with 5
.mu.M ascorbic acid, 100 U/ml penicillin and 100 ug/ml
streptomycin. This process was carried out to wash away the
cryopreservatives in which the osteoclast cells had been
frozen.
[0054] The resulting osteoclast precursor cell suspension was
counted using a hemacytometer to ascertain the number of osteoclast
precursor cells/ml. An aliquot of cell suspension containing a
total of two million osteoclast cells was removed and added to the
15 ml centrifuge tube containing the six million osteoblast cells.
The volume of medium in the centrifuge tube was then was adjusted
to a total of 10 ml by the addition of fresh 10% FBS-DMEM
supplemented with 5 .mu.M ascorbic acid, 100 U/ml penicillin and
100 ug/ml streptomycin. Finally, the 10 ml of medium containing
both osteoblast and osteoclast cells was supplemented with 50 ng/ml
macrophage colony stimulating factor (M-CSF) and 50 ng/ml of
receptor activator of NF-kB (RANK) ligand.
[0055] The resulting osteoblast/osteoclast cell suspension was then
inoculated into a 10 ml rotating cell culture system (RCCS) flask
(also know as a High Aspect Ratio Vessel--HARV) (Synthecon, Inc.)
and horizontally rotated at 2 RPM for a period of 24 hr to allow
coalescence of the osteoblast and osteoclast cells into a solid,
three dimensional tissue construct. After a period of 24 hr, the
rotation speed of the HARV was increased to 14 RPM in order ensure
that the tissue construct was maintained in an optimal position
within the HARV, namely not touching or hitting the sides of the
rotating HARV rather in a state of "free-fall" within the medium
contained within the rotating HARV. The cell medium within the HARV
was exchanged with 10 ml of fresh 10% FBS-DMEM supplemented with 5
.mu.M ascorbic acid, 100 U/ml penicillin, 100 ug/ml streptomycin,
50 ng/ml macrophage colony stimulating factor (M-CSF) and 50 ng/ml
of receptor activator of NF-kB (RANK) ligand (a matrix-free culture
medium) after every fourth day of culture.
[0056] After a period of seven days of culture in the HARV under
the above conditions the medium was exchanged for 10 ml of fresh
10% FBS-DMEM supplemented with 5 .mu.M ascorbic acid, 100 U/ml
penicillin, 100 ug/ml streptomycin, 50 ng/ml macrophage colony
stimulating factor (M-CSF), 50 ng/ml of receptor activator of NF-kB
(RANK) ligand, 200 .mu.M hydrocortisone-21-hemisuccinate and 10 mM
beta-glycerophosphate (a matrix-free mineralization culture
medium). The hydrocortisone-21-hemisuccinate and
beta-glycerophosphate were added to the medium to induce
mineralization of the tissue construct by the osteoblasts. The cell
medium within the HARV was exchanged with 10 ml of fresh 10%
FBS-DMEM supplemented with 5 .mu.M ascorbic acid, 100 U/ml
penicillin, 100 ug/ml streptomycin, 50 ng/ml macrophage colony
stimulating factor (M-CSF), 50 ng/ml of receptor activator of NF-kB
(RANK) ligand, 200 .mu.M hydrocortisone-21-hemisuccinate and 10 mM
beta-glycerophosphate every fourth day until the tissue construct
was harvested.
Example 3
Imaging of Bone Constructs
[0057] The method of Example 2 was followed, with the following
differences: primary osteoblasts and osteoclast precursors were
mixed together at about a 2:1 ratio of osteoblasts to osteoclast
precursors, with the total number of cells being about 9 million
cells; the mixture of cells was then horizontally rotated at 2 rpm
for 48 hrs, and then at 14 rpm for 5 days; and mineralization
proceeded at 16 rpm for 21 days. The resulting mineralized
three-dimensional bone constructs are pictured in FIG. 2A (at 14
days of mineralization) and FIG. 2B (at 21 days of mineralization).
The scale bar in each figure is 1 cm.
Example 4
Bone Constructs with Labeled Osteoclasts
[0058] The method of Example 2 was followed, with the following
differences: osteoclast precursors were labeled with the
fluorescent CellTracker-Red probe (Invitrogen, Inc.) prior to
mixing with osteoblasts; primary osteoblasts and labeled osteoclast
precursors were mixed together at about a 2:1 ratio of osteoblasts
to osteoclast precursors, with the total number of cells being
about 3 million cells; the mixture of cells was then horizontally
rotated at 2 rpm for 24 hrs, and then at 14 rpm for 5 days; and
mineralization proceeded at 16 rpm for 14 days. FIG. 3A shows a
fluorescence confocal microscopy image of an optical section
through some of the resulting mineralized three-dimensional bone
constructs. The results show that osteoclast precursor cells
(observable as white spots in FIG. 3A) have spatially arranged
themselves as an outer layer of the mineralized three-dimensional
bone constructs with the putative osteoblast cells being embedded
in the crystalline matrix of the central region of the constructs.
FIG. 3B shows the same constructs viewed in incident laser light
(i.e. non-fluorescent illumination) to illustrate the shape of the
constructs. The scale bar in each of FIG. 3A and FIG. 3B is 200
.mu.m.
Example 5
Optical Sectioning of a Bone Construct with Labeled Osteoclasts
[0059] The method of Example 2 was followed, with the following
differences: osteoclast precursors were labeled with the
fluorescent CellTracker-Green probe (Invitrogen, Inc.) prior to
mixing with osteoblasts; primary osteoblasts and labeled osteoclast
precursors were mixed together at about a 2:1 ratio of osteoblasts
to osteoclast precursors, with the total number of cells being
about 6 million cells; the mixture of cells was then horizontally
rotated at 2 rpm for 48 hrs, and then at 14 rpm for 5 days; and
mineralization proceeded at 16 rpm for 21 days. Three dimensional
reconstruction of a resulting large bone construct was performed
using Z series confocal imaging. Panels A-I in FIG. 4 are the
individual images used by the confocal imaging software to build
the optical reconstruction of the bone construct in three
dimensions, each image representing a sequential view over the
surface of the construct (white spots indicate individual cells).
FIG. 4 indicates that the arrangement of osteoclasts to the outer
layer of the construct remains a feature of the construct even
after extended culture periods (i.e. a total of four weeks in the
HARV vessel, including three weeks grown in mineralization
conditions). Labeled osteoclasts are apparent in an outer layer
covering the surface of the construct. Additionally, structures
reminiscent of resorption pits or lacunae found in actively
remodeling bone in vivo can be clearly seen on the surface of the
OsteoSphere in Panel J of FIG. 4 (indicated by arrows, Bar equals
300 microns).
Example 6
Staining of Bone Constructs with Alizarin Red S, von Kossa, and
Harris Hematoxylin Stains
[0060] Mineralized three-dimensional bone constructs were prepared
as detailed in Example 3. The bone constructs were then fixed using
a Bouin's solution (a rapid penetrating fixative solution), frozen
sectioned, and stained for calcium using the Alizarin red S stain
and for phosphates and carbonates using the von Kossa histochemical
stain. FIG. 5A shows a 5.times. magnification image of Alizarin red
S staining and FIG. 5B shows a 20.times. magnification image of
Alizarin red S staining (which appears as the dark regions of the
images). FIG. 5C shows a 5.times. magnification image of von Kossa
staining and FIG. 5D shows a 20.times. magnification image of von
Kossa staining (which appears as the dark regions of the images).
The results demonstrate that the crystalline matrix of the
mineralized three-dimensional bone constructs contain mineral
elements observed in normal human bone in vivo. In addition, when a
composite, low-power image of a complete 10 micron thick frozen
cross-section of a Bouin's fixed OsteoSphere stained with Alizarin
red S was generated (FIG. 5E), an external zone (indicated by
arrows) surrounding the OsteoSphere could be clearly discerned (Bar
equals 500 microns). This outer zone surrounded the mineralized
internal core of the OsteoSphere. This external zone of the
OsteoSphere had been previously determined to contain osteoclast
cells determined by confocal microscopy imaging as described in
Examples 4 and 5 and shown in FIGS. 3 and 4.
[0061] The same sections were also stained for the presence of
nucleated cells using the Harris Hematoxylin stain. The results are
shown in FIG. 6A (5.times. magnification image) and FIG. 6B
(20.times. magnification image). The dark regions of the image
indicate staining. The staining pattern illustrates a large number
of cells embedded within the crystalline matrix of the three
dimensional construct. These cell nuclei appear intact with little
or no signs of nuclear fragmentation, a histological indicator of
the occurrence of cell death/apoptosis. Arrows in FIG. 6B point to
large numbers of cells embedded within the crystalline matrix in
the three dimensional construct. Cell nuclei appear intact with
little or no signs of nuclear fragmentation; such fragmentation
would be a histological indicator of the occurrence of cell
death/apoptosis. Immuno-staining of these sections for the presence
of osteoblast cell markers, such as alkaline phosphatase, indicated
the absence of osteoblast cell markers in the cell type embedded in
the crystalline matrix. Thus, it is believed that the cells
embedded in the crystalline matrix are osteocytes.
Example 7
Detection of Osteocalcein, an Osteoblast Differentiation Marker, in
Bone Constructs Using Immunofluorescence
[0062] The method of Example 2 was followed, with the following
differences: osteoclast precursors were labeled with the
fluorescent CellTracker-Orange probe (Invitrogen, Inc.) prior to
mixing with osteoblasts; primary osteoblasts and labeled osteoclast
precursors were mixed together at about a 2:1 ratio of osteoblasts
to osteoclast precursors, with the total number of cells being
about 6 million cells; the mixture of cells was then horizontally
rotated at 2 rpm for 48 hrs, and then at 14 rpm for 5 days; and
mineralization proceeded at 16 rpm for 21 days. The resulting
mineralized three-dimensional bone constructs were fixed using a
phosphate buffered saline solution (pH 7.2) containing 1% (v/v)
freshly generated formaldehyde. The fixed bone constructs were then
immunochemically stained using a monoclonal antibody against
osteocalcein (an osteoblast differentiation marker) as the primary
antibody and an Alexa 488-labeled secondary antibody. FIG. 7A-7C
shows images obtained by simultaneously imaging both markers in one
of the bone constructs using confocal microscopy. Specifically,
FIG. 7A shows osteocalcein staining, FIG. 7B shows
CellTracker-Orange staining, and FIG. 7C shows the same construct
illuminated with incident laser light. The results indicate that
osteocalcein staining and CellTracker-Orange staining (both visible
as a white "ring" around the construct in FIGS. 7A and 7B) are
spatially localized to the same area of the construct. This
indicates that the osteoclast precursor cells are localized to the
same region as differentiated mature osteoblasts and that both were
spatially localized to the surface of the construct.
Example 8
Demonstration of Bone Morphogenic Protein (BMP) Production by
OsteoSpheres
[0063] The method of Example 6 was followed for producing frozen
sections of Bouin's fixed, mineralized OsteoSpheres grown for 21
days under mineralization conditions. A total of eight, 10 micron
frozen sections of Bouin's fixed mineralized OsteoSpheres were
collected and total RNA was extracted from the material using a
micro-scale mRNA extraction/purification kit. The presence of
intact mRNA in the extract was verified using a Pico.TM. Total mRNA
Chip Assay (Agilent Technologies). The OsteoSphere-derived mRNA was
then converted to cDNA and duplicate samples of cDNA where then
probed with human sequence primer sets directed against sequences
of either 18S ribosomal RNA (control), BMP-2, BMP-4 or BMP-7 using
a real-time quantitative PCR assay (BioRad Laboratories). FIG. 8
demonstrates the expression of both BMP-2 and BMP-7 mRNA by
OsteoSpheres as detected using a real-time quantitative PCR.
Specifically, Panel A of FIG. 8 demonstrates that mature, 21 day
old mineralized OsteoSpheres produced approximately eight times
more BMP-2 mRNA than BMP-7 mRNA as indicated by CT values
("crossing the threshold"--horizontal line labeled CT, FIG. 8A) of
37 cycles for BMP-2 and approximately 40 cycles for BMP-7. In
contrast, the CT value for the 18S ribosomal RNA control is
approximately 25. No significant amount of BMP-4 mRNA was detected
in the 21 day old mineralized OsteoSphere sample. Analysis of the
melt curve generated for the assay indicates that the appropriate
sized amplicons had been generated in the RT-qPCR assay (Panel
B).
Example 9
Generation of an HLA-Negative Osteogenic Precursor Cell Line for
Use in the Production of Three Dimensional Human Bone
Constructs
[0064] The ideal bone graft material for use in long bone
fractures/nonunions/defects and spinal fusion procedures is a
material that has all of the characteristics of an autogenous bone
graft without the need for a second surgical procedure to harvest
the material. In addition, the material should preferably contain
all those elements necessary to provide the optimal cellular
environment for new bone formation and the promotion of fusion with
the existing matrix of the bone being treated. By providing a bone
graft material having these elements, bony fusion will occur in a
predictable and efficient manner reducing the time required for the
patient to heal.
[0065] Development of mineralized three-dimensional bone constructs
(sometimes referred to as "OsteoSpheres") using, for example, the
methods disclosed in the preceeding examples, demonstrates that
bone material similar to autogenous bone graft with both
osteoinductive and osteoconductive properties can be generated
using an ex vivo approach. As such, this approach can be used to
produce autologous bone graft material using osteoblasts (harvested
from normal healthy bone) and osteoclasts (harvested from
peripheral blood lymphocytes) for a single patient. However, this
autologous transplantation approach has limited utility for bone
graft procedures in general. Although adult human bone is
considered an immune privileged tissue under certain circumstances
(i.e. exampled by the incorporation of cadaveric bone during
allograft transplantation), transplantation of OsteoSpheres that
contain living osteoblast, osteoclast and osteocyte cell types into
a non-tissue type matched individual may induce a host-graft
reaction.
[0066] Allograft bone material contains a variety of cellular
material derived from dead cells contained in the mineral matrix
(including a variety of growth factors and Bone Morphogenetic
Proteins (BMPs)) but no living cells. The osteoconductive
properties of allograft material center on the ability of the
allograft to promote infiltration of the three dimensional
mineralized matrix with osteogenic cells from the existing bone of
the patient. These cells colonize the three dimensional matrix of
the allograft and begin to incorporate the graft material into the
existing bone matrix. During this process osteoclasts and
osteoblasts infiltrate the allograft material; osteoclasts break
down the mineralized allograft material and osteoblasts form new
mineralized matrix until the allograft material has been fully
integrated into the existing bone. This process can take several
years to complete and the rate and efficiency of the incorporation
is dependent on a variety of factors including the amount of BMP's
found in the allograft and the availability of osteogenic cells
from the patient's existing bone.
[0067] During this incorporation process, demineralization of the
allograft material by infiltrating osteoclasts does not release any
living cells into the recipient, therefore preventing a host-graft
immune response. However, in the case of OsteoSpheres used for bone
graft purposes, the possibility does exist that living cells (i.e.
osteocytes, osteoblasts or osteoclasts) contained within the
OsteoSpheres when released into the recipient's bone could initiate
a host-graft response; therefore this may represent a significant
barrier to the use of OsteoSpheres in non-autologous or non-tissue
matched bone graft procedures. However, it is entirely possible
that due to the slow rate and the small numbers of such "foreign"
cells released at any given time from the mineralized matrix during
incorporation of OsteoSpheres, a clinically significant host-graft
immune response may not be mounted by the patient.
[0068] One approach to eliminate any possibility of a host-graft
reaction is to produce OsteoSpheres using osteoblast and osteoclast
cells that are not recognized as being foreign by the recipient's
immune system. One means of achieving this goal is to develop human
osteoblast and osteoclast pre-cursor cell lines that are incapable
of generating a host-graft immune response due to the lack of human
lymphocyte antigen (HLA) expression by these cells. As osteoblasts
and osteoclasts are terminally differentiated cells, creation of
such cell lines directly from these terminally differentiated cells
may prove difficult due to the limited proliferative capacity of
such cells.
[0069] Another approach to achieving the goal of osteoblast and
osteoclast cell lines incapable of expressing HLA is to develop an
osteogenic precursor cell line derived from embryonic or
mesenchymal stem cells which are incapable of HLA expression. These
cells can then in turn be differentiated into non-HLA expressing
osteoblasts and osteoclasts for use in producing non-HLA expressing
OsteoSpheres for bone grafting applications. Such osteogenic
differentiation of stem cells has previously been achieved by
exposing these cells to osteogenic differentiation factors such as
BMP's. However to date, such simple exposure of stem cells to
selected differentiation factors in tissue culture has resulted in
varied levels of efficiency with regard to the numbers of cells
that progress along the desired differentiation pathway.
[0070] A much more rapid and higher differentiation rates of stem
cells may be achieved by mimicking the cellular milieu of the
tissue to which differentiation is being driven. For example, human
embryonic or mesenchymal stem cells that are grown on an irradiated
feeder layer of murine bone marrow cells exhibit better osteogenic
differentiation rates than those exposed to selected BMPs. However,
exposure to such feeder layers poses significant contamination
risks for stem cells destined for human clinical applications.
Recent studies have shown that exposure of human embryonic stem
cells to complex, soluble protein extracts of target tissues such
as lung, drives differentiation towards the lung cell phenotype
more efficiently. In addition, the presence of a membrane
permeabilizing agent, polyethylene glycol, in the tissue culture
medium significantly enhanced this response. These data suggest
that optimal "driving" of the differentiation process towards a
particular target tissue may be more efficient if the cells are
cytoplasmically loaded with a mixture of complex proteins extracted
from the cellular milieu to which they are being targeted.
[0071] Even if osteogenic differentiation can be enhanced to
produce large numbers of human osteoblasts and osteoclasts, the
issue surrounding the possibility of a host-graft response after
implantation cannot be ignored. As mentioned above, one approach to
overcoming the problem is the development of a stem cell line,
either embryonic or adult, that has been genetically modified so as
to not express the major proteins involved in "self-foreign"
recognition at the cellular level, namely the human lymphocyte
antigen family of cell surface proteins. Several approaches have
previously been used for silencing of HLA expression in a variety
of cell types. These include the introduction of RNA interference
cassettes containing short hair-pin RNA (shRNA) sequences that
target the gene encoding for the HLA Class 1 antigen. This approach
has been demonstrated in a variety of cells including peripheral
blood lymphocytes and human embryonic cells using lentivirus or
adenovirus vectors to transfect the sequence into the recipient
cells. The incorporation and transduction of these shRNA sequences
by the transfected cell generates inhibitory RNA (RNAi) sequences
that in turn block or "knock down" the HLA Class 1 expression in
these cells without affecting the expression of other cellular
proteins. Although this approach indicates that stable transfection
of cells with specific inhibitory RNA sequences (RNAi) can be used
as a means of silencing HLA expression, the use of lentivirus and
adenoviral transfection vectors raises many safety concerns with
regard to the potential neoplastic properties of these virally
infected cells.
[0072] A related approach to silencing a particular gene is the
production of "knock-out" cells that have been injected with
partial DNA sequences corresponding to the gene of interest. This
approach is used to generate "knock-out" or transgenic rodents by
microinjection of partial or mutated DNA sequences directly into
the nuclei of the cells of the blastocyst. These injected
blastocysts are then implanted into females and allowed to gestate.
During cell division, the partial DNA sequences recombine with the
gene of interest, resulting in some cases in daughter cells from
which this "mutated" gene has been excised from the genome. This
process is random and does not always result in excision of the
target gene. In those cases where excision does occur without
inducing lethal disruption of the genome, the resulting surviving
off-spring have cells with a normal genome absent the targeted gene
and are referred to as "knock-out" animals for this particular
gene.
[0073] A similar "knock-out" approach has been suggested for
producing mesenchymal stem cells (MSC's) extracted from adult
tissues such as bone marrow. These MSC's exhibit multi-potent
properties and can be induced to undergo differentiation into a
range of cell and tissue types, including bone and cartilage.
Targeted disruption of gene expression in this fashion, such as the
HLA family of genes, provides a possible means of generating an HLA
negative MSC line that can be propagated indefinitely in culture
and guided down the osteogenic cell differentiation pathway at will
by exposing them to specific differentiation factors.
[0074] One of the persistent technical problems in producing either
"knock-out" or "silenced" embryonic or adult stem cells has been
the means of delivering the required genetic material to the cell.
Use of plasmid constructs containing viral promoter sequences has
raised many ethical issues concerning the subsequent use of such
genetically modified cells in human clinical applications. In
addition, chemical or irradiation-induced site mutagenesis raises
the possibility of unintended collateral damage of the genome
leading to unforeseen complications in these cells, such as the
development of a neoplastic phenotype in vivo. As such, a means of
delivering the required genetic material to the stem cell without
the need to expose the cells to potentially harmful methods and/or
reagents greatly enhances the possibility of using these
genetically modified cells in human clinical applications.
[0075] Recently there has been described a highly efficient means
of transferring a wide variety of macromolecules, such as dextrans,
proteins, antibodies and plasmid DNA, directly into living cells
without the need for exposure of the recipient cells to any
chemical or viral vectors (U.S. Pat. No. 6,221,666). This
technology, known as impact-mediated loading, is based on the
concept that entry of large macromolecules into the cell cytoplasm
that are normally impermeant to the cell plasma membrane, can be
achieved by generating transient, survivable disruptions or
"membrane wounds" of the plasma membrane. Such membrane wounds are
a normal constitutive cellular response to mechanical loading of a
variety of cell types in the body (i.e. cardiac myocytes, skeletal
muscle myofibers, epithelial and endothelial cells) and provide a
route through which a macromolecule can directly enter the cell
cytoplasm from the extra-cellular fluid by diffusion. However, such
constitutive membrane wounds are normally small and provide limited
communication between the extra-cellular environment and the cell
cytoplasm.
[0076] By developing the impact loading technique and apparatus for
producing reproducible membrane wounds of relatively uniform size
in a controlled fashion, gaining direct access to the cell
cytoplasm became routinely possible. In addition, we also
demonstrated that the efficiency of this transfer process (in terms
of overall cell survival, the amount of macromolecule transferred
and the size of the macromolecule that can be transferred) is
significantly improved if the impact-mediated loading process is
carried out under hyper-gravity conditions. Furthermore, unlike a
variety of other techniques used for direct transfer of protein or
genetic material into the living cell cytoplasm, this technique is
applicable to all cell types including primary cells of both human
and animal origin, producing significantly higher yields of loaded
cells in a shorter period of time than competing technologies.
[0077] For example, this technique has been used to load a variety
of macromolecules including large proteins, IgG antibodies and
inert dextrans (up to 2.times.10.sup.6 daltons in size) directly
into primary human cells. The technique has also been used to
achieve transfection of primary human cells with plasmid DNA
constructs normally resistant to such transfer protocols. The
impact-mediated loading technique allows large numbers of cells
(>10,000 cells) to be simultaneously loaded in a matter of
seconds, with cell survival rates approaching 95%-100% of the
starting cell population depending on the loading conditions; the
smaller the molecule being loaded the higher overall
loading/transfection efficiency that can be achieved. As RNAi
sequences and partial DNA sequences are relatively small in terms
of their molecular size, these compounds are ideally suited for
delivery using the impact-mediated loading technology. As such,
this technique is ideally suited for the production of genetically
modified human embryonic stem cells without the need for the use of
chemical or viral transfection agents that may modify the cells in
unwanted or unanticipated ways.
[0078] Described herein is a method for producing a human embryonic
stem cell line or an adult stem cell line of mesenchymal origin in
which at a minimum HLA Class I protein expression has been
silenced: (1) by the stable expression of an inhibitory RNA
sequence for the human HLA Class I gene (HLA1-RNAi) contained in a
shRNA cassette transfected into these cells; or (2) as a
consequence of HLA gene "knock-out" by delivery of a partial DNA
sequence of the HLA gene of interest Inhibitory RNAi sequences for
HLA Class I, shRNA transfection cassettes and the DNA sequence for
the HLA Class I gene have previously been described in the
literature. Such RNAi sequences, partial DNA sequences and shRNA
cassettes are now available from a variety of commercial sources in
purified form. These cells may also be transfected with similar
shRNA cassettes containing the inhibitory RNA sequences for
additional human HLA genes or other "self-foreign" recognition
genes such as beta-microglobulin. Transfection of embryonic or
adult stem cells may be achieved by loading the cells with purified
shRNA cassettes encoding for these RNAi sequences to HLA genes
using the impacted mediated loading technology known as The
G-Loader. The G-loader device itself is approximately 25 cm tall
and 10 cm in diameter (see FIG. 8). A 35 mm tissue culture plate
containing a monolayer of adherent cells is placed in the base of
the G-Loader after being incubated with loading medium containing
the macromolecule of interest. Prior to placing the cell sample
into the G-Loader, a particle cartridge is inserted into the device
consisting of a circular cartridge that supports a rupturable
membrane on which is located a layer of 10 micron particles
arranged in a specific pattern (see panel insert). In addition, the
device has been charged with air to the required pressure and the
on-board g-load accelerometer trigger has been set to the required
g-value using the G-Loader Charging Station (for bench top
operation there is a manual trigger button located on the side wall
of the device). When the G-Loader is activated, either manually or
by reaching a set g-load a metered volume of pressurized air is
directed into the particle cartridge, the membrane ruptures and
disperses the particles into the air stream. The particles then
impact the cell layer inducing membrane wounding in a high
controllable and reproducible fashion.
[0079] FIG. 10 shows enhanced impact-mediated loading under
hypergravity conditions. Specifically, FIG. 10 shows fluorescent
micrographs of Swiss 3T3 cell monolayers cytoplasmically loaded
with Fluor.TM. 488 goat-anti-mouse IgG employing impact-mediated
loading at 1.times.g on the bench-top (Panel B) and at 200.times.g
in a bench top centrifuge (Panel C). Control cells (Panel A) were
exposed to IgG for the same period of time but were not impact
loaded. Panels A, B and C were taken at the same photographic
conditions. Note the larger amounts of IgG present in the cytoplasm
of cells loaded at 200.times.g as compared to those loaded at
1.times.g and that IgG is excluded from the nuclear regions of the
loaded cells.
[0080] FIG. 11 shows fluorescent micrographs of primary human
skeletal myoblasts (HSKMC), Swiss 3T3 cells (3T3) and primary
bovine capillary endothelial cells (BCEC) 24 hr after
impact-mediated loading of a plasmid construct encoding for a green
fluorescent protein, Lantern Green.TM. (pLG).
[0081] Alternatively, DNA sequences can be directly transferred to
the cell nuclei using standard microinjection techniques.
Transfection efficiency using the impact-mediated loading technique
may be optimized in these cell types by mechanistically selecting
the optimal impact velocity, size of the impact particle and the
optimal g-load at which to perform the transfer. The transfer
process may also be enhanced by pre-treatment of the cell surface
prior to loading with naturally-derived hyaluronidase enzyme and/or
a low molecular weight surfactant such as PF-68 to increase access
of the purified genetic material to the cell membrane surface. In
addition, co-loading of low molecular weight surfactants, such as
PF-68, appear to enhance transfection efficiency by a mechanism
postulated to involve encapsulation of the genetic material in a
protective layer of surfactant thereby inhibiting intracellular
degradation prior to expression. Such an approach may also increase
the degree of random transfer of partial DNA sequences to the cell
nuclei from the cytoplasm of loaded cells.
[0082] After transfection with either RNAi sequences or partial DNA
sequences for HLA genes, cells are returned to their standard
culture conditions for a period of three days. At this time, the
loaded cell population are cloned by limiting dilution and the
resulting clones are again expanded in culture until such time that
enough cells have been generated in order to test for the
expression of specific RNAi, RNA or DNA sequences in extracts of a
portion of the cloned cell population. Depending on the genetic
material delivered to the original cells, these clones are also
tested for: (1) the presence of the HLA RNAi sequence, and (2) the
absence of RNA encoding the HLA gene; or (3) the absence of the DNA
sequence encoding the HLA gene. Those clones showing evidence of
(1) HLA RNAi sequence expression and a lack of HLA gene RNA
expression or (2) the lack of HLA gene DNA sequences, are further
expanded in tissue culture while cryogenically preserving portions
of the cloned cell population.
[0083] Following a suitable period of time to allow expansion of
the cloned cells, a sample of cells from the surviving clones is
exposed to tissue culture medium containing osteoblastic
differentiation agents, such as ascorbic acid,
beta-phosphoglycerol, hydrocortisone-21-hemisuccinate, BMP-2, BMP-4
and/or BMP-7, for a period of seven days and then tested for the
production osteoblastic cell markers such as alkaline phosphatase
and osteocalcin. In addition, a sample of these cloned cells is
also exposed to tissue culture medium containing osteoclastic
differentiation agents (i.e. RANKL, M-CSF) and then tested for the
production of osteoclastic cell markers such as fluoride-sensitive
tartrate-resistant acidic phosphatase (TRAP).
[0084] As an alternative means of driving osteogenic cell
differentiation by simple exposure of the cloned stem cells to
various selected compounds dissolved in their tissue culture
medium, cloned stem cells are also cytoplasmically loaded with a
complex soluble protein extract of primary human osteoblast or
osteoclast cultures using the impact mediated loading technology.
The cells are then cultured for a further seven days in culture and
tested for osteoblastic and osteoclastic markers as detailed
above.
[0085] Depending on the genetic material delivered to the original
cells from which these clones were derived, the differentiated
cells are also tested for the absence of either DNA encoding the
HLA gene or mRNA encoding HLA proteins or for the presence of RNAi
sequences directed against the HLA genes. Criteria for selecting
cloned cells for further expansion include the following: (1)
positive for the HLA RNA sequence and negative for HLA mRNA or (2)
negative for the DNA HLA gene sequence; and (3) the ability to
differentiate into osteoblasts or osteoclasts depending on the
differentiation stimulus provided.
[0086] Those clones that are capable of differentiating into
osteoblasts and/or osteoclasts are further expanded in order to
develop a cell line in which HLA expression has been silenced by
stable transfection of RNAi sequences against the HLA gene(s) or by
HLA gene knockout. These cells are then differentiated into
osteoblasts and osteoclasts and combined using the methods of the
preceeding examples to produce OsteoSpheres for clinical use in
bone graft procedures. Cell material is extracted from fully
mature, mineralized OsteoSpheres. Depending on the genetic material
delivered to these cells, this material is tested for the
expression of HLA RNAi sequences and the absence of HLA mRNA or the
absence of the HLA DNA gene sequence. In addition, all samples are
tested by protein immuno-blotting for the absence of actual HLA
protein in order to determine that the constituent cells of the
OsteoSpheres are indeed negative for HLA expression at both the
genetic and protein levels.
* * * * *